Bombyx Mori Nucleopolyhedrovirus Research Articles

The cytoplasmic tail substitution increases the assembly efficiency of Ebola virus glycoprotein on the budded virus of Bombyx mori nucleopolyhedrovirus

Glycoprotein (GP1,2) of the Ebola virus (EBOV) is the key membrane fusion protein, which is a key candidate protein for vaccine preparations. Previously, GP1,2 was expressed by Bombyx mori nucleopolyhedrovirus (BmNPV) expression vector system; however, few GP1,2 was incorporated into budded virus (BV) of BmNPV. To improve the incorporation efficiency of GP1,2 into the virion, the GP1,2 fusion with the cytoplasmic tail of GP64 of BmNPV was expressed in BmN cells by the BmNPV expression system. The BV was purified by ultracentrifugation, and GP1,2 expression in BV was detected by the antibody. The result indicated that a 532% increase in the relative GP1,2 densitometry signal was observed in constructs utilizing the GP64 C-terminal domain; moreover, the substitution of GP1,2 native signal peptide with GP64 signal peptide increased the incorporation efficiency by 34.6% in the relative GP1,2 densitometry signal. We revealed that the application of the cytoplasmic tail of BmNPV GP64 significantly increased the incorporation rate of GP1,2 into the BV envelope. This study lays a foundation for GP1,2 vaccine development.

The Bombyx mori Nucleopolyhedrovirus GP64 Retains the Transmembrane Helix of Signal Peptide to Contribute to Secretion across the Cytomembrane.

ABSTRACTBombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms that causes severe economic losses in sericulture. GP64 is the key membrane fusion protein that mediates budded virus (BV) fusion with the host cell membrane. Previously, we found that the n-region of the GP64 signal peptide (SP) is required for protein secretion and viral pathogenicity; however, our understanding of BmNPV GP64 remains limited. Here, we first reported that BmNPV GP64 retained its SP in the mature protein and virion in only host cells but did not retain in nonhost cells. Uncleaved SP mediates protein targeting to the cytomembrane or secretion in Bombyx mori cells. The exitance of the n-region extended the transmembrane helix length, which resulted in the cleavage site to be located in the helix structure and thus blocked cleavage from signal peptidase (SPase). Without the n-region, the protein fails to be transported to the cytomembrane, but this failure can be rescued by the cleavage site mutation of SP. Helix-breaking mutations in SP abolished protein targeting to the cytomembrane and secretion. Our results revealed a previously unrecognized mechanism by which SP of membrane fusion not only determines protein localization but also determines viral pathogenicity, which highlights the escape mechanism of SP from the cleavage by SPase.IMPORTANCE BmNPV is the primary pathogen of silkworms, which causes severe economic losses in sericulture. BmNPV and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are closely related group I alphabaculoviruses, but they exhibit nonoverlapping host specificity. Recent studies suppose that GP64 is a determinant of host range, while knowledge remains limited. In this study, we revealed that BmNPV GP64 retained its SP in host cells but not in nonhost cells, and the SP retention is required for GP64 secretion across the cytomembrane. This is the first report that a type I membrane fusion protein retained its SP in mature proteins and virions. Our results unveil the mechanism by which SP GP64 escapes cleavage and the role of SP in protein targeting. This study will help elucidate an important mechanistic understanding of BmNPV infection and host range specificity.

Mutations in the polyhedrin NLS affect the assembly and polyhedral shape of alphabaculovirus occlusion bodies

Alphabaculoviruses produce occlusion bodies (OBs) in the nucleus of the infected cells at the late stage of infection. OBs are mainly composed of a single viral protein called polyhedrin (POLH). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) POLH possesses a monopartite nuclear localization signal sequence (NLS), KRKK, from 32nd to 35th residues. However, the functions of POLH NLS of other alphabaculoviruses remain unknown. Here, POLH NLS mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated and NLS function as well as the relationship between NLS and OB localization or morphology was investigated. Deletion or mutation of BmNPV POLH NLS severely affected POLH and OB intracellular localization. Additionally, viruses in which the arginine residue at the 33rd position of POLH was mutated produced a lower number of OBs, which was presumably due to decreased POLH accumulation in the infected cells. Furthermore, cytoplasmic OBs were morphologically aberrant, even though nuclear OB morphology was normal in the same cell. These results indicate that NLS is required for nuclear localization and efficient accumulation of BmNPV POLH, which heavily affect the number and morphology of OBs.

Potential Proteins Interactions with Bombyx mori Nucleopolyhedrovirus Revealed by Co-Immunoprecipitation.

Simple SummaryThe Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the major pathogens of the silkworm. We reconstituted the virus in vitro and used it as a bait for immunoprecipitation experiments on cells and silkworm bodies, obtaining a database of proteins potentially interacting with BmNPV. This study provides a protein data reference for the screening of BmNPV receptors and the study of proteins that play a key role in the replication of BmNPV. It is also important to deal with the prevention of BmNPV in silkworms at the molecular level.Virus–host interactions are critical for virus replication, virulence, and pathogenicity. The Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the most common and harmful pathogens in sericulture. Herein, we used co-immunoprecipitation to identify candidate proteins with potential interactions with BmNPV. First, a recombinant BV virus particle rBmBV-egfp-p64-3×flag-gp64sp was constructed using a MultiBac baculovirus multigene expression system. Co-immunoprecipitation experiments were then performed with the recombinant BV virus infected with BmN cells and Dazao silkworms. LC-MS/MS analysis revealed a total of 845 and 1368 candidate proteins were obtained from BmN cells and silkworm samples, respectively. Bioinformatics analysis (Gene Ontology, KEGG Pathway) was conducted for selection of proteins with significant enrichment for further confirmation of the effects on BmNPV replication. Overall, the results showed that SEC61 and PIC promoted the replication of BmNPV, while FABP1 inhibited the replication of BmNPV. In summary, this study reveals the potential proteins involved in BmNPV invasion and proliferation in the host and provides a platform for identifying the potential receptor proteins of BmNPV.

Utilization of Recombinant Baculovirus Expression System to Produce the RBD Domain of SARS-CoV-2 Spike Protein.

Continuous outbreaks of viral diseases in humans facilitates a need for the rapid development of viral test kits and vaccines. These require expression systems to produce a pure and high yield of target viral proteins. We utilized a baculovirus–silkworm expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. First, we had to develop a strategy for constructing a recombinant baculovirus for RBD expression. For this, the coding region of the Bombyx mori cypovirus (BmCPV) polyhedron was assembled with the Bombyx mori nuclear polyhedrosis virus (BmNPV) promoter. We demonstrated that the recombinant baculovirus has the ability to form polyhedrons within host silkworm cells. In addition, the encapsulated BVs are able to infect silkworms by ingestion and induce foreign protein expression. In this way, we utilized this novel system to obtain a high yield of the target foreign protein, the RBD of the SARS-CoV-2 S protein. However, the viral infection rate of our recombinant BV needs to be improved. Our study shed light on developing a highly efficient expression system for the production of antigens and subsequent immunoassays and vaccines.

Analysis of Silver Nanoparticles for the Treatment and Prevention of Nucleopolyhedrovirus Affecting Bombyx mori.

Bombyx mori nucleopolyhedrovirus (BmNPV) causes major economic losses in sericulture. A number of agents have been employed to treat viral diseases. Silver nanoparticles (AgNPs) have wide applications in biomedical fields due to their unique properties. The anti-BmNPV effect of AgNPs has been evaluated, however, there are insufficient studies concerning its toxicity to other organisms and the environment. We chemically synthesized biocompatible BSA-AgNPs with a diameter range of 2–4 nm and characterized their physical properties. The toxicity of AgNPs towards cells and larvae with different concentrations was examined; the results indicated a biofriendly effect on cells and larvae within specific concentration ranges. The SEM observation of the surface of BmNPV after treatment with AgNPs suggested that AgNPs could destroy the polyhedral structure, and the same result was obtained by Coomassie blue staining. Further assays confirmed the weakened virulence of AgNPs-treated BmNPV toward cells and larvae. AgNPs also could effectively inhibit the replication of BmNPV in infected cells and larvae. In summary, our research provides valuable data for the further development of AgNPs as an antiviral drug for sericulture.

Metabolic characterization of hemolymph in Bombyx mori varieties after Bombyx mori nucleopolyhedrovirus infection by GC-MS-based metabolite profiling.

The "Huakang 2" silkworm variety, bred by the Sericulture ResearchInstitute of the Chinese Academy of Agricultural Sciences, is highly resistant to Bombyx mori nucleopolyhedrovirus (BmNPV) and effectively solves the issue of frequent Bombyx mori nuclear polyhedrosis in sericultural production. The molecular mechanism of its resistance to BmNPV, however, is still unknown. The purpose of the present study was therefore to identify these anti-BmNPV mechanisms by using metabolomics in combination with transcriptomics after subcutaneous injection of budded virus (BV) with high concentrations of BmNPV from specimens of the Baiyu N variety (which is highly resistant to BmNPV) and the Baiyu variety (which is sensitive to BmNPV). A total of 375 differential metabolites were identified, which mainly included sugars, acids, amines, alcohols, glycosides, and other small molecules. KEGG enrichment analysis and functional clustering of differential metabolites identified possible metabolic pathways, including tyrosine metabolism, oxidative phosphorylation, and alanine, aspartate, and glutamate metabolism. The differentially expressed genes (DEGs) identified by transcriptome analysis were annotated in KEGG. Association analysis showed that the metabolic pathways of different silkworm varieties are affected differently by BmNPV infection, triggering a series of complex physiological and biochemical changes in the organism. In particular, oxidative phosphorylation might be an essential pathway involved in regulation of disease resistance.

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.

Establishment of a Novel Baculovirus-Silkworm Expression System.

The baculovirus vector expression system is a well-established tool for foreign protein production and gene delivery. In this study, we constructed a recombinant baculovirus vector system. The UAS promotor region and Bombyx mori nucleopolyhedrovirus (BmNPV) polyhedrin coding region were ligated into a pFastBac Dual vector to obtain a BmBac-UPS recombinant bacmid. The recombinant bacmid BmBac-Gal4 was generated by the same strategy which has a Gal4 coding region controlled by the IE2 promoter. BmBac-UPS and BmBac-IGal4 were co-infected into silkworm BmN cells to confirm the ability of the UAS/Gal4 system to form polyhedrons in B. mori cells. Furthermore, the recombinant viruses were tested for infection efficiency and the ability to generate polyhedra in transgenic B. mori cell line BmE. The results showed that recombinant viruses have the ability to form polyhedrons and gain raised pathogenicity when orally infected B. mori larvae and are applied as the preferred tool for foreign gene delivery and expression

Identification and Characterization of Genes Related to Resistance of Autographa californica Nucleopolyhedrovirus Infection in Bombyx mori.

Simple SummaryAutographa californica nucleopolyhedrovirus (AcMNPV) is a kind of baculovirus that was initially found and named for its host, but the previous study reveals several silkworm strains are preferentially susceptible to AcMNPV through intrahemocelical injection method. In the following study, genetics analysis showed that a set of potential genes which controlled resistance of AcMNPV was located on chromosome 3. In the present research, we performed Genome-Wide Association Studies to identify the gene that controls the resistance of AcMNPV, results show that the Niemann-Pick C1 (NPC-1) gene is strongly associated with this resistance. Then we found that there are several amino acid mutations in the protein sequence of BmNPC1 between two different resistance strains of Bombyx mori. RNAi results showed that BmNPC1 successfully suppressed virus infection ability and changed the expression pattern of viral genes.In Bombyx mori, as an important economic insect, it was first found that some strains were completely refractory to infection with Autographa californica nucleopolyhedrovirus (AcMNPV) through intrahemocelical injection; whereas almost all natural strains had difficulty resisting Bombyx mori nucleopolyhedrovirus (BmNPV), which is also a member of the family Baculoviridae. Previous genetics analysis research found that this trait was controlled by a potentially corresponding locus on chromosome 3, but the specific gene and mechanism was still unknown. With the help of the massive silkworm strain re-sequencing dataset, we performed the Genome-Wide Association Studies (GWAS) to identify the gene related to the resistance of AcMNPV in this study. The GWAS results showed that the Niemann-Pick type C1 (NPC-1) gene was the most associated with the trait. The knockdown experiments in BmN cells showed that BmNPC1 has a successful virus suppression infection ability. We found a small number of amino acid mutations among different resistant silkworms, which indicates that these mutations contributed to the resistance of AcMNPV. Furthermore, inhibition of the BmNPC1 gene also changed the viral gene expression of the AcMNPV, which is similar to the expression profile in the transcriptome data of p50 and C108 strains.

Dual display hemagglutinin 1 and 5 on the surface of enveloped virus-like particles in silkworm expression system

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform.

β-Arrestin 2 acts an adaptor protein that facilitates viral replication in silkworm

β-Arrestin 2 is known to be a widely distributed adaptor protein in mammals but its function has never been reported in Lepidoptera insects. Herein, the β-Arrestin 2 (BmArrestin 2) gene from silkworm was cloned and characterized. The spatiotemporal expression level of BmArrestin 2 was highest in the gonads at the 3rd day of 5th instar, whereas the highest and lowest abundance of BmArrestin 2 were identified in the tracheal and testis, respectively. BmArrestin 2 is mainly distributed in the cytoplasm. Furthermore, in BmN cells，overexpression of BmArrestin 2 promoted Bombyx mori nucleopolyhedrovirus (BmNPV) and B. mori cytoplasmic polyhedrosis virus (BmCPV) replication as the increment of the concentration of plasmid transfection, whereas silencing the gene with specific siRNA inhibited viral replication. Replication of BmNPV and BmCPV also was weakened using BmArrestin 2 antiserum as the increment of the concentration. Immunofluorescent staining revealed the invasion of recombinant BmNPV or BmCPV was decreased after blocking endogenous BmArrestin 2. On the other hand, BmArrestin 2 co-localizes with recombinant BmNPV and BmCPV virions in BmN cells. These results suggest that BmArrestin 2 may represent a novel target for antiviral strategies, as it is an adaptor protein that plays a key role in virus replication.

Identification and Characterization of BmNPV m6A Sites and Their Possible Roles During Viral Infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens and causes serious economic losses in sericulture. At present, there is no epigenetic modification of BmNPV transcripts, especially of m6A, and this modification mediates diverse cellular and viral functions. This study showed that m6A modifications are widespread in BmNPV transcripts in virally infected cells and the identified m6A peaks with a conserved RRACH sequence. m6A sites predominantly appear in the coding sequences (CDS) and the 3′-end of CDS. About 37% of viral genes with m6A sites deleted from the viral genome did not produce any infectious virions in KOV-transfected cells. Among the viral genes related to replication and proliferation, ie-1 mRNA was identified with a higher m6A level than other viral genes. The m6A sites in the ie-1 mRNA may be negatively related to the protein expression. Viral replication was markedly inhibited in cells overexpressed with BmYTHDF3 in a dose-dependent manner, and a contrary effect was found in si-BmYTHDF3-transfected cells. Collectively, the identification of putative m6A modification in BmNPV transcripts provides a foundation for comprehensively understanding the viral infection, replication, and pathobiology in silkworms.

Identification of key metabolic pathways reprogrammed by BmNPV in silkworm Bombyx mori

Elucidating the mechanism of infection of Bombyx mori nuclear polyhedrosis virus (BmNPV) and host antiviral response remains a major scientific task in sericulture. Virus invasion causes a series of antiviral immune responses in the host, and successful infection leads to massive changes in the host’s physiological and biochemical state. Current research mainly focuses on silkworm genes and proteins associated with viral infection and resistance, but little is known regarding the host metabolic pathways that the virus utilizes for optimal replication. In this work, key metabolites involved in viral infection were identified, including trehalose, riboflavin, tryptophan, tyrosine, and phenylalanine. The genes associated with metabolite biosynthesis and catabolism were analyzed, and their expression levels were found to be largely consistent with their respective metabolite levels before and after viral treatment in both strains. The screened metabolites were further investigated for their roles in viral replication using exogenous metabolite addition into the culture medium. The results showed that tryptophan effectively inhibited BmNPV replication, while glutamine promoted viral replication in a dose-dependent manner. Trehalose and riboflavin exhibited a complex effect on BmNPV replication. This study outlines the critical metabolites and metabolic pathways required for BmNPV to proliferate and infect the host, indicting the potential of metabolite-based treatment for viral inhibition.

Identification of endoplasmic-reticulum-associated proteins involved in Bombyx mori nucleopolyhedrovirus entry by RNA-seq analysis

Membrane fusion is a key step in enveloped virus infection, releasing the viral genome into the cytoplasm to initiate infection. Bombyx mori nucleopolyhedrovirus (BmNPV) is an enveloped DNA virus that mainly infects silkworms. Information about membrane fusion of BmNPV with host cells is still limited. In this study, BmN cells were pretreated with ??ammonium chloride??, and infection with BmNPV was allowed to occur naturally through endocytosis or artificially through low-pH-induced fusion with the plasma membrane, after which the cells were subjected to RNA-seq. The results indicated that a few endoplasmic reticulum-associated proteins (ERAPs) were among the common upregulated DEGs, including BiP, CRT, and HSP90, and this upregulation was confirmed by q-PCR. Knockdown of BiP, CRT, and HSP90 expression by siRNA resulted in significant inhibition of BmNPV infection. This study suggests that ERAPs may be involved in the BmNPV membrane fusion process during infection.

BmNPV p35 Reduces the Accumulation of Virus-Derived siRNAs and Hinders the Function of siRNAs to Facilitate Viral Infection.

Antiviral immunity involves various mechanisms and responses, including the RNA interference (RNAi) pathway. During long-term coevolution, viruses have gained the ability to evade this defense by encoding viral suppressors of RNAi (VSRs). It was reported that p35 of baculovirus can inhibit cellular small interference RNA (siRNA) pathway; however, the molecular mechanisms underlying p35 as a VSR remain largely unclear. Here, we showed that p35 of Bombyx mori nucleopolyhedrovirus (BmNPV) reduces the accumulation of virus-derived siRNAs (vsiRNAs) mapped to a particular region in the viral genome, leading to an increased expression of the essential genes in this region, and revealed that p35 disrupts the function of siRNAs by preventing them from loading into Argonaute-2 (Ago2). This repressive effect on the cellular siRNA pathway enhances the replication of BmNPV. Thus, our findings illustrate for the first time the inhibitory mechanism of a baculovirus VSR and how this effect influences viral infection.

Cellular Lnc_209997 suppresses Bombyx mori nucleopolyhedrovirus replication by targeting miR-275-5p in B. mori.

Long non-coding RNA (lncRNA) is a type of non-coding RNA molecule, which exceeds 200 nucleotides in length and participates in the regulation of a variety of life activities. Recent studies showed that lncRNAs play important roles in viral infection and host immunity. At present, the researches on insect lncRNAs are relatively few. In this study, we found the expression of Lnc_209997 was significantly down-regulated in silkworm fat body infected with Bombyx mori nucleopolyhedrosis virus (BmNPV). Inhibition of Lnc_209997 promoted BmNPV replication. Enhancing the expression of Lnc_209997 inhibited the proliferation of BmNPV. miR-275-5p was up-regulated in silkworm fat body infected with BmNPV. Dual luciferase reporter gene system confirmed the interaction between Lnc_209997 and miR-275-5p. Over-expression of Lnc_209997 inhibited the expression of miR-275-5p, while inhibition of Lnc_209997 enhanced the expression of miR-275-5p. Further, over-expression of miR-275-5p can facilitate the replication of BmNPV. These results suggested that BmNPV could increase the expression of miR-275-5p by inhibiting cellular Lnc_209997 expression to promote their own proliferation. Our results are helpful for better understanding the role of lncRNAs in BmNPV infection, and provide insights into elucidating the molecular mechanism of interaction between Bombyx mori and virus.

Humoral immune response induced with dengue virus-like particles serotypes 1 and 4 produced in silkworm

Dengue is an arboviral disease, which threatens almost half the global population, and has emerged as the most significant of current global public health challenges. In this study, we prepared dengue virus-like particles (DENV-LPs) consisting of Capsid-premembrane-envelope (CprME) and premembrane-envelope (prME) polypeptides from serotype 1 and 4, which were expressed in the silkworms using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid. 1CprME, 1prME, 4CprME, and 4prME expressed proteins in hemolymph, and the molecular weight of the purified proteins was 55 kDa, respectively. The purified polypeptides formed spherical Dengue virus-like particles (DENV-LPs) with ~ 30–55 nm in diameter. The immunoelectron microscopy (IEM) images revealed antigens to the surface of a lipid bilayer of DENV-LPs. The heparin-binding assay shows a positive relationship between absorbance and E protein domain III (EDIII) quantity, which is supported by the isothermal titration calorimetry assay. This indicates a moderate binding affinity between heparin and DENV-LP. The high correlation between patient sera and DENV-LP reactivities revealed that these DENV-LPs shared similar epitopes with the natural dengue virus. IgG elicitation studies in mice have demonstrated that DENV-LPs/CPrMEs elicit a stronger immune response than DENV-LP/prMEs, which lends credence to this claim.

SUMOylation Regulates BmNPV Replication by Moderating PKIP Intracellular Localization

SUMOylation is a reversible covalent process between a small ubiquitin-like modifier (SUMO) and its target protein and has become a crucial regulator of protein functions. Here, we report that Bombyx mori nucleopolyhedrovirus (BmNPV) may take advantage of the host SUMOylation system to enhance its own replication, similar to many other viruses. Both the knockdown of BmSUMO by RNAi and chemical blocking by ginkgolic acid both impaired BmNPV replication. Using site mutation and pull-down assays, we found that lysine K70 of the protein kinase-interacting protein (PKIP), which is conserved in all Alphabaculoviruses, was modified by SUMO. Mutation of K70 in PKIP led to its translocation from the cytoplasm to the nucleus. Knockout and rescue experiments showed that the rescue of PKIP mutant virus with wild-type PKIP restored BmNPV replication to the normal level, but this was not true for the K70R mutation. Altogether, these results show that SUMOylation of PKIP plays a key role in BmNPV replication.

Acetylation of late expression factor 4 is crucial for the transcription and proliferation of Bombyx mori nucleopolyhedrovirus

Late expression factor 4 (LEF4), RNA polymerase subunit of Bombyx mori nucleopolyhedrovirus (BmNPV), plays an enzymatic role to enhance the capping of pre-mRNA of late and very late genes. Lysine acetylation is apost-translational modification process having many important functions associated with the regulation of agene expression. Our previous study on lysine acetylome in BmNPV infected BmN cells showed that LEF 4 was acetylated at lysine 76 (K76). However, it is still unclear whether the modification of K76 residue contributes to the modulation of viral gene transcription. To elucidate the role played by acetylation or deacetylation of LEF4 K76 in the transcription of viral genes, we constructed acetylation mimicking and deacetylation mimicking mutant virus, K76Q and K76R, respectively. We then transfected BmN cells with these mutants and analyzed the level of pre-mRNA at different times. The K76R showed asignificant decrease in the mRNA transcription level of vp39 and p10 genes at 48 and 72h post-transfection, while K76Q did not show any significant changes compared with lef4-Wt. We further detected the virus titer of lef4-Wt, K76Q [et] K76R, and it was found that K76R impaired the virus infectivity ability at 72 and 96h, while K76Q did not affect the virus infectivity. Moreover, the yeast two hybrid technique (Y2H) showed that both mutants (K76Q [et] K76R) affected the association of LEF 4 with the P47 protein. Taken together, these results indicated that acetylation modification of K76 is important for the proper transcription of late and very late genes, and the effectiveness of viral infection. Keywords: BmNPV lef4 gene; lysine acetylation, late genes transcription; BmNPV p47 gene; infectivity.